Alzheimer's disease (AD) is characterized by the excessive generation and accumulation of p-amyloid peptides (Ap). y-secretase, the enzyme responsible for immediate release of Ap, is one of the most important drug targets in AD therapies. Most current y-secretase inhibitors lack discrimination between y-cleavage of APP and other substrates including Notch, and show severe toxicity after chronic administration in animals. We have shown that y-secretase generation of Ap in an N2a cell-free system is ATP dependent. In addition, Gleevec, an Abl kinase inhibitor, which acts by competing at the ATP-binding site of this tyrosine kinase, potently reduces Ap production. Gleevec also reduces Ap production in rat primary neuronal cultures and in vivo in guinea pig brains. However, Gleevec does not inhibit y-secretase-catalyzed cleavage of Notch-1. Our recent results suggest that ATP analogues and Gleevec enhance the binding of holo-APP and APP-CTF to presenilin-1. Furthermore, Gleevec retards egress of APP- but not Notch-containing vesicles from the ER in a PS1-dependent manner. More importantly, we have identified a novel Gleevec binding protein (GBP). We hypothesize that Gleevec achieves its actions on trafficking and cleavage of APP through this novel GBP, and propose specific experiments to test this hypothesis. One goal is to use Gleevec and other ATP analogues as tools to investigate the molecular steps involved in Ap formation. This should prove of enormous value in Alzheimer's disease research. To achieve this goal, in our revised application, we will characterize the effects of ATP analogues and Gleevec on kinetics, substrate binding, and conformational changes of y-secretase (Aim I); we will characterize the selective effects of Gleevec on trafficking of APP vs Notch (Aim II); we will examine the action of a newly discovered Gleevec binding protein (GBP) on the modulation of y-secretase activity and APP trafficking (Aim III); we will analyze the effects of Gleevec on ADrelated pathology, electrophysiology and behavior, using AD transgenic mice (Aim IV); we will characterize the mechanism by which inhibitor 2, another Abl kinase inhibitor, which also acts by competing at the ATPbinding site of this tyrosine kinase, modulates y-secretase activity (Aim V). Taken together, these studies should elucidate the underlying mechanism by which Gleevec regulates y-secretase activity. Our study should accelerate the development of novel therapeutic strategies against Alzheimer's disease.